For flow cytometric analysis, the mesenchymal stem cells (MSCs) at passage three were trypsinized, centrifuged at 300 g for 8 min, and resuspended in phosphate-buffered saline (PBS) at a concentration of 1 × 106 cells/mL. A total of 100 µL of aliquots were labeled for 30 min with antibodies against CD14, CD45 (fluorescein isothiocyanate (FITC)), or CD73; CD34 phycoerythrin (PE; Becton Dickinson, USA); or CD106, CD29 PE, or CD44 (FITC; Becton Dickinson), washed with 1 mL of stain buffer (BD-Pharmingen, USA) and resuspended in 500 µL of stain buffer. The labeled cells were analyzed using an argon-ion laser with a wavelength of 488 nm (FACS Calibur; Becton Dickinson). A total of 10,000 events were obtained and analyzed with the DIVA software program (Becton Dickinson). Control staining with appropriate isotype-matched monoclonal antibodies was included.
Diva software program
Manufactured by BD
Sourced in Italy
The DIVA software program is a powerful tool designed for the acquisition, analysis, and management of flow cytometry data. It serves as a versatile platform for researchers and scientists working in various fields, including immunology, cell biology, and clinical diagnostics. The software's core function is to provide a comprehensive and user-friendly interface for the collection, processing, and interpretation of flow cytometry data.
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2 protocols using diva software program
1
Characterization of Mesenchymal Stem Cells by Flow Cytometry
2
Immunophenotyping of T Helper Cells
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Mononuclear cells were treated for 30 min at room temperature with commercially available FITC-, PE-, or PE-Cy5-conjugated monoclonal antibody (mAb): anti-CD4 (Becton-Dickinson Biosciences, Italy), anti-IL23R (R&D Systems Inc., Minneapolis, MN, USA), and anti-IL-17 (R&D Systems); anti-CD161 (Becton-Dickinson Biosciences, Italy). FITC-, PE-, or PE-Cy5-conjugated isotype-matched mouse mAb was used to set the fluorescence background (IgG1 and IgG2a, BD Pharmigen). Appropriate isotype controls and fluorescence minus one (FMO) controls were used to assign gates. Expression of the cytoplasmic cytokine was evaluated as previously described [15] . CD4+ T cells were gated with two different approaches: physical characteristic of cells and expression of CD4 in the area of lymphocytes to identify the T helper cells. Fluorescence-activated cell sorter (FACS) analysis was assessed as previously described, briefly, 5 × 105 cells were acquired on FACSCalibur 6 analyzer (Becton-Dickinson) and data processed by DIVA software program (Becton-Dickinson Biosciences, Italy). To evaluate whether differences between peaks were statistically significant with respect to controls, the Kolmogorov-Smirnov test was used.
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